Peptides mimicking the biological activity of steroid hormones and their uses

ABSTRACT

A monoclonal antibody to a steroid hormone is used for the isolation from a combinatorial peptide library of a peptide mimicking the biological activity of said steroid hormone. Peptides having estrogenic-like and progestational-like activity were synthesized and assayed for their ability to compete with the respective steroid hormone for binding to the monoclonal antibody and for binding to said steroid hormone receptor. Peptides having estrogenic-like activity were found to be selective to estrogen receptor alpha or beta and thus may have more clinical applications than the non-selective steroid hormone estradiol.

FIELD OF THE INVENTION

The present invention relates to peptides mimicking the biologicactivity of steroidal hormones, to methods for their identification andisolation and pharmaceutical compositions comprising them.

-   Abbreviations: BSA—bovine serum albumin; CK—creatine kinase;    E2—estradiol; ER—estrogen receptor; mAb—monoclonal antibody;    PBS—phosphate-buffered saline; RIA—radioimmunoassay;

BACKGROUND OF THE INVENTION

Estradiol, a steroid hormone, regulates the growth, differentiation andfunction of diverse tissues, both within and outside the reproductivesystem. Because of the multiple target organs (e.g. heart, uterus,brain, breast, immune cells, etc) for estrogens and the occurrence ofboth beneficial and unwanted effects during treatment, the key toimprovement in drug therapy is the development of selective estrogenreceptor modulators (SERM's) with better tissue selectivity (Warner etal., 1999). Estrogen mediates its effects via the estrogen receptor (ER)that exists as two subtypes, ER α (Greene and Press, 1986) and ER β(Kuiper et al., 1996; Kuiper and Gustafsson, 1997; Nillson, 1998), whichdiffer in the C-terminal domain and in the N-terminal transactivationdomain. The two ER isoforms exhibit distinct tissue distributionpatterns and differ in their ligand binding ability andtransactivational properties (Kuiper et al., 1997).

In the technique of phage-displayed peptide library, a diversecollection of random peptides displayed on the surface of filamentousphages can be screened for phages that bind to a target molecule, suchas an antibody or receptor. This method generates phagotopes that haveconsensus sequences often matching the sequence found in the peptidicantigen used for generating the antibody used as the selector molecule.The advantages of phage displayed peptide libraries for drug discoveryhave been reported (Devlin J. J., 1990; Lowman, 1997). Numerous groupsused these peptide libraries to identify short peptide mimetics ofantigenic epitopes (Cortese et al., 1994; Scott and Smith, 1990),ligands for receptors (Balass et al., 1993; Cabilly et al., 1998a;Cabilly et al., 1998b; Yayon et al., 1993), for proteins (egstreptavidin) (Giebel, 1995) and for biotin binders (Saggio, 1993).Phage peptide libraries and other combinatorial peptide libraries havebeen extensively utilized in the last years for mapping antigenicepitopes using as probes monoclonal antibodies (mAb) to proteinicantigens.

Although phage displayed peptide libraries have been widely used tostudy protein-protein interactions (Cesareni et al., 1999; DeWitt,1999), the use of phage peptide library to understand the interaction ofproteins with compounds of non-peptidic nature has been limited.Exceptions to this approach are studies related to peptide that miniccarbohydrate antigens (Moe et al., 1999; Qiu et al., 1999). To ourknowledge, combinatorial peptide libraries have not been used to isolatepeptides mimicking the activity of small molecular weight non-peptidicorganic compounds such as steroids.

SUMMARY OF THE INVENTION

The guiding principle of the present invention is based on theassumption that the binding site of monoclonal antibodies (mAb)recognizing a given steroid represents a molecular template for thesteroid molecule. Thus when one finds peptides which recognize such amAb and compete with the steroid for the mAb template, such peptidesmight mimic the steroid in its molecular structure as well as in itsbiological characteristics.

The detection of such peptides is facilitated by the use of suitablesynthetic or biological libraries. When a steroid receptor is available,the interaction of the lead peptides with their homologous receptor isinvestigated. Final test of the lead peptide is naturally carried out bytheir action in vivo in mice or other suitable animals.

It is thus the purpose of the present invention to provide peptides,hereinafter “the peptides of the invention”, that mimic the biologicalactivity of steroid hormones.

The present invention thus provides a synthetic peptide that mimics thebiological activity of a steroid hormone, selected from:

(i) a peptide exhibiting a steroid hormone-like biological activity;

(ii) a peptide obtained from (i) by deletion of one or more amino acidresidues;

(iii) a peptide obtained by addition to a peptide (i) or (ii) of one ormore natural or non-natural amino acid residues;

(iv) a peptide obtained by replacement of one or more amino acidresidues of a peptide (i) to (iii) by the corresponding D-stereomer, byanother natural amino acid residue or by a non-natural amino acidresidue;

(v) a chemical derivative of a peptide (i) to (iv);

(vi) a cyclic derivative of a peptide (i) to (v);

(vii) a dual peptide consisting of two of the same or different peptides(i) to (vi), wherein the peptides are covalently linked to one anotherdirectly or through a spacer; and

(viii) a multimer comprising a number of the same or different peptides(i) to(vi).

In one embodiment, the steroid hormone is an estrogen such as estradiol(17β-estradiol), estrone and estriol and the peptide of the inventionhas estrogenic-like activity.

In another embodiment, the steroid hormone is a progestogen such asprogesterone and the peptide of the invention has progestational-likeactivity.

In a further embodiment, the steroid hormone is an androgen such astestosterone and the peptide of the invention has androgenic-likeactivity.

In still a further embodiment, the steroid hormone is a corticoid suchas cortisone, hydrocortisone and corticosterone and the peptide of theinvention has adrenocorticoid-like activity.

The present invention further provides novel conjugates of theestrogenic peptides of the invention for detection of estrogen receptorson tumor cells, particularly breast cancer cells. The conjugates may beformed with fluorescent markers or with chelating agents such aspentetic acid (DTPA) and either particles of a paramagnetic element suchas gadolinium or of a radioactive element such as In⁺⁺⁺. In addition,the invention further provides novel conjugates of the estrogenicpeptides of the invention with chemotherapeutic drugs such as adriamycinand daunomycin, for affinity targeting and treatment ofestrogen-sensitive tumors, particularly breast cancer.

In another aspect, the present invention relates to the use of amonoclonal antibody to a steroid hormone for the isolation from acombinatorial peptide library, of a peptide exhibiting said steroidhormone biologic-like activity.

The present invention further provides a method for screening acombinatorial peptide library for the identification of a peptideexhibiting a steroid hormone biologic-like activity, which comprises:

(i) providing a monoclonal antibody (mAb) with high affinity andspecificity to the steroid hormone investigated;

(ii) screening a combinatorial peptide library with said monoclonalantibody of (i) for the identification of peptides that bindspecifically to said mAb;

(iii) isolating said peptides and testing them for competition with saidsteroid hormone for binding to said mAb in vitro and for binding to saidsteroid hormone receptor in vitro; and

(iv) identifying the peptides that mimic said steroid hormone activityas being those that successfully compete with said steroid hormone forbinding to its respective mAb and receptor.

The invention further provides pharmaceutical compositions comprising apharmaceutically acceptable carrier and a peptide of the invention.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows dose response curves for estradiol (squares) and theestrogen-like peptides 1 (triangles) and 2 (circles) in a competitivebinding radioimmunoassay (RIA).

FIG. 2 shows dose response curves for estradiol (squares) and theestrogen-like peptides 1 (triangles) and 2 (circles) using a receptorbinding assay for estrogen receptor (ER) alpha.

FIG. 3 shows dose response curves for estradiol (squares) and theestrogen-like peptides 1 (triangles) and 2 (circles) using a receptorbinding assay for ER beta.

FIG. 4 shows dose response curves for progesterone (circles) and theprogestational-like peptides 3 (triangles) and 4 (squares) in acompetitive binding radioimmunoassay (RIA).

DETAILED DESCRIPTION OF THE INVENTION

According to the invention, the possibility of employing combinatorialpeptide libraries was explored to identify peptides that display steroidhormone-like activity. Such an approach offers dual advantage; first,from the structural point of view, it is of interest to test whether asteroid, a hydrophobic polycyclic non-water soluble compound, can bemimicked by a water soluble peptide. Second, if it does, from theapplication point of view, this allows the preparation of biasedcombinatorial libraries of chemical and biological origin.

According to the present invention, it is possible to develop peptideswith selective activity, for example peptides selective to ER α and/orER β with appropriate tissue selectivity profiles. The selectivity shownherein for the peptides having estrogenic-like activity is a veryimportant characteristic and advantage of such peptides of theinvention, particularly vis-à-vis estradiol itself that is notselective.

In one embodiment of the present invention, we used a specific mAb toestradiol, clone 15, prepared years ago in the laboratory of one of theinventors, to screen a phage-displayed random peptide library toidentify a 15-mer peptide that displays estrogenic-like activity in bothantibody and receptor binding assays in vitro and in causing an increasein the specific activity of creatine kinase (CK) in rat tissues in vivo.CK was chosen as a response marker since we have previously shown thatCK can be used as a response marker for estrogenic activity in vivo inrat animal models and in vitro in skeletal cells (Somjen, 1998; Somjenet al., 1996). Moreover, CK is related to changes in cell division andcan be used as a marker for the interaction of estradiol with thefunctional estrogen receptor (Malnick, 1983).

Thus, this 15-mer peptide exhibiting estrogenic-like activity, initiallycalled H5 and herein in the specification and claims identified aspeptide 1, was isolated from a 15-mer phage-peptide library withanti-estradiol mAb clone 15, then sequenced and synthesized by standardmethods. An extended version of the peptide 1 was then synthesized andconverted to the cyclic peptide 2.

Peptides 1 and 2 have the following sequences:Leu-Pro-Ala-Leu-Asp-Pro-Thr-Lys-Arg-Trp-Phe- (1) Phe-Glu-Thr-Lys Cyclic[Cys-Ala-Glu-Leu-Pro-Ala-Leu-Asp-Pro- (2)Thr-Lys-Arg-Trp-Phe-Phe-Glu-Thr-Lys-Pro-Pro- Pro-Pro-Cys]

Both peptides 1 and 2 were then tested in competitive assays withestradiol for the binding sites of the clone 15 and for binding with theestradiol receptor, thus finding that they bind to the estradiolreceptor alpha but not to estradiol receptor beta. In vivo, the peptidesincreased the specific activity of creatine kinase in some rat tissueswhile estradiol caused an increase in all rat tissues.

Peptide 1 was then shortened at the N- and/or C-terminal to givepeptides with shorter sequence which bind mAb-15 and ER. Then, one ormore amino acid residues of the sequences were replaced by differentresidues thus obtaining the peptides presented in Tables 3 to 5 inExample 8 hereinafter. These peptides were evaluated in terms of bindingto anti-estradiol mAb clone 15 and some of them were evaluated also interms of binding to ER α and β. From the results obtained with thesepeptides it can be concluded that peptides having estrogenic-likeactivity derived from peptide 1 should have at least 6 amino acidresidues and contain the core peptide WFX₁E wherein X₁ is F or Y.

Thus, in one preferred embodiment, the invention relates to a peptidehaving estrogenic-like activity of at least 6 amino acid residues of thesequence:X₄-X₂-Trp-Phe-X₁-Glu-X₃wherein

-   -   X₁ is Phe or Tyr;    -   X₂ is Lys-Arg-, Ala-Arg-, Lys-Ala-, Val-Arg-, Lys-Pro-,        Val-Ser-, or Ile-Arg-;    -   X₄ is hydrogen or Thr-, Pro-Thr-, Asp-Pro-Thr-,        Leu-Asp-Pro-Thr-, Ala-Leu-Asp-Pro-Thr-,        Pro-Ala-Leu-Asp-Pro-Thr-, Leu-Pro-Ala-Leu-Asp-Pro-Thr-, or        Cys-Ala-Glu-Leu-Pro-Ala-Leu-Asp-Pro-Thr-; and    -   X₃ is hydroxyl, Thr, -Thr-Lys, or -Thr-Lys-Pro-Pro-Pro-Pro-Cys;        and cyclic derivatives thereof.

In preferred embodiments, the anti-estrogenic peptide is selected fromthe peptides herein designated peptides 1, 2, 44, A-43, A-44, 39, 21,B34 and B37 of the sequences:Leu-Pro-Ala-Leu-Asp-Pro-Thr-Lys-Arg-Trp-Phe- (1) Phe-Glu-Thr-Lys Cyclic[Cys-Ala-Glu-Leu-Pro-Ala-Leu-Asp-Pro- (2)Thr-Lys-Arg-Trp-Phe-Phe-Glu-Thr-Lys-Pro-Pro- Pro-Pro-Cys]Lys-Arg-Trp-Phe-Phe-Glu (44) Ala-Arg-Trp-Phe-Phe-Glu (A43)Lys-Ala-Trp-Phe-Phe-Glu (A44) Val-Arg-Trp-Phe-Phe-Glu (39)Lys-Pro-Trp-Phe-Phe-Glu (21) Val-Ser-Trp-Phe-Phe-Glu (B34)Ile-Arg-Trp-Phe-Phe-Glu (E37)

It is to be understood that any combinatorial peptide library can beused according to the invention. The examples herein show the use ofphage-displayed random peptide library but other libraries such ascombinatorial synthetic peptide libraries can be used.

According to the invention, once a peptide that binds the monoclonalantibody is identified, it is then synthesized by standard peptidesynthesis methods and tested in vitro in competitive assays with thesteroid hormone for the binding sites of the respective monoclonalantibody and for the receptor, and then in vivo for testing the steroidhormone-like activity.

Peptides of the invention having progestational-like activity include,but are not limited to, 15-mer and 16-mer peptides, analogs, derivativesand cyclic forms thereof such as, for example, the peptides hereinidentified as peptides 3 and 4 of the sequences:Val-Asn-His-Pro-Trp-Asp-Gln-Ala-Gln-Phe-Leu-Ser-Thr-Ile  (3)Ser-Asn-Pro-Phe-Cys-Gln-Thr-Asp-Gly-Asp-Cys-His-Val-His-Thr  (4)

As mentioned above, the term “peptides of the invention” as used hereinincludes: (i) synthetic peptides having the biological activity of asteroid hormone such as those disclosed herein; (ii) peptides obtainedfrom (i) by deletion of one or more amino acid residues; (iii) peptidesobtained by addition to peptides (i) or (ii) of one or more natural ornon-natural amino acid residues; (iv) peptides obtained by replacementof one or more amino acid residues of peptides (i) to (iii) by thecorresponding D-stereomer, by another natural amino acid residue or by anon-natural amino acid residue; (v) chemical derivatives of the peptides(i) to (iv); (vi) cyclic derivatives of peptides (i) to (v); (vii) dualpeptides consisting of two of the same or different peptides (i) to(vi), wherein the peptides are covalently linked to one another directlyor through a spacer; and (viii) multimers comprising a number of thesame or different peptides (i) to(vi), as long as the peptides (ii) to(viii), also herein referred to sometimes by the terms “peptidederivative” or “peptide analogue”, present substantially the samebiological activity of the parent peptide (i).

Typically, modifications are made that retain the steroid hormone-likeactivity of the parent peptide (i). Any of the above modifications maybe utilized alone or in combination, provided that the modified sequenceretains the steroid hormone-like activity of the parent peptide,identified by means of the appropriate assays.

Deletion of amino acid residues or addition of one or more natural ornon-natural amino acid residues may be made at the N- or the C-terminalof the parent peptide. Substitutions include replacement of the naturalamino acid residues by the corresponding D-amino acid residue, forexample to increase blood plasma half-life of a therapeuticallyadministered peptide, or by different natural amino acid residues or bynon-natural amino acid residues. Thus, the peptide or peptide derivativeof the invention may be all-L, all-D or a D,L-peptide.

A “chemical derivative” of a peptide of the invention includes, but isnot limited to, a derivative containing additional chemical moieties notnormally a part of the peptide provided that the derivative retains thesteroid hormone function of the peptide. Examples of such derivativesare: (a) N-acyl derivatives of the amino terminal or of another freeamino group, wherein the acyl group may be either an alkanoyl group,e.g. acetyl, hexanoyl, octanoyl, or an aroyl group, e.g. benzoyl; (b)esters of the carboxy terminal or of another free carboxy or hydroxygroups; (c) amides of the carboxy terminal or of another free carboxygroups produced by reaction with ammonia or with a suitable amnine,resulting in the C-terminus or another carboxy group being in the form—C(O)—NH—R, wherein R may be hydrogen, C1-6 alkyl, such as methyl,ethyl, propyl, isopropyl, butyl, isobutyl, pentyl or hexyl, aryl such asphenyl, and aralkyl such as benzyl, such amidation being advantageous inproviding additional stability and possibly enhanced activity to thepeptide; (d) glycosylated derivatives; (e) phosphorylated derivatives;(f) derivatives conjugated to lipophilic moieties e.g. caproyl, lauryl,stearoyl; and (g) derivatives conjugated to an antibody or othercellular ligands. Also included among the chemical derivatives are thosederivatives obtained by modification of the peptide bond —CO—NH—, forexample by (a) reduction to —CH₂—NH—; (b) alkylation to —CO—N (alkyl)-;(c) inversion to —NH—CO—.

The term “cyclic peptides” as used herein refers to cyclic derivativescontaining either an intramolecular disulfide bond, i.e. —S—S—, anintramolecular amide bond, i.e. —CONH— or —NHCO—, or intramolecularS-alkyl bonds, i.e. —S—(CH₂)_(n)—CONH— or —NH—CO(CH₂)_(n)—S—, wherein nis 1 or 2. The cyclic derivatives containing an intramolecular disulfidebond may be prepared by conventional solid phase synthesis whileincorporating suitable S-protected cysteine or homocysteine residues atthe positions selected for cyclization such as the amino and carboxyterminals of the peptides, with the option of including spacingresidues, such as (Ala)_(n), (Gly)_(n) where n is from 1 to 4, ornon-natural amino acids such as 6-aminocaproic acid, between theterminal residue and the linking residue. The linking residues may thenbe linked together using known techniques to form cyclicized peptidederivatives. For example, the peptide 2 was prepared by elongation fromlinear peptide 1 and cyclized according to methods known in the art forformation of a disulphide bond. Following completion of the chainassembly, cyclization can be performed either by selective removal ofthe S-protecting groups with a consequent on-support oxidation of freecorresponding two SH-functions, to form S—S bonds, followed byconventional removal of the product from the support and appropriatepurification procedure, or by removal of the peptide from the supportalong with complete side-chain deprotection, followed by oxidation ofthe free SH-functions in highly dilute aqueous solution. The cyclicderivatives containing an intramolecular amide bond may be prepared byconventional solid phase synthesis while incorporating suitable aminoand carboxyl side-chain protected amino acid derivatives at thepositions selected for cyclization. The cyclic derivatives containingintramolecular —S-alkyl bonds may be prepared by conventional solidphase synthesis while incorporating an amino acid residue with asuitable amino-protected side chain, and a suitable S-protected cysteineor homocysteine residue at the positions selected for cyclization.Cyclic peptides may be prepared also as backbone cyclic peptides asdescribed in the literature (Gilon et al., 1991). A backbone cyclizationis a method developed to impose conformational constraints on peptidesby interconnecting the peptide backbone atoms (N or C^(α)) to eachother, to side chains, or to amino and carboxy terminals (Bitan et al.,1997; Gilon et al., 1991)

A “dual peptide” according to the invention consists of two the same ordifferent peptides or peptide derivatives of the invention covalentlylinked to one another directly or through a spacer such as by a shortstretch of alanine residues or by a putative site for proteolysis bycathepsin (see U.S. Pat. No. 5,126,249 and European Patent No. 495,049with respect to such sites). This will induce site-specific proteolysisof the preferred form into the two desired analogues.

“Multimers” according to the invention consist of polymer moleculesformed from a number of the same or different peptides or derivativesthereof. The polymerization is carried out with a suitablepolymerization agent, such as 0.1% glutaraldehyde (Audibert et al.(1981) Nature, 289: 593)

The peptides of the invention, in addition to mimicking the steroidhormone activity, may form the basis of novel pharmaceutical agentsproviding significant advantages over currently available steroid drugssuch as estrogenic and anti-estrogenic drugs. Since these peptides, incontrast to steroid hormones, contain reactive amino acids such as alysine group, novel pharmaceutical agents can be obtained by conjugationto markers, e.g. fluorescent molecules, paramagnetic particles, orradioactive tags, for use in prognosis of treatment of hormone-dependentcarcinomas, or to chemotherapeutic drugs such as adriamycin ordaunomycin, for reduction of the amount of the toxic antineoplasticagent in cancer treatment.

In endocrine therapy of cancers with hormone receptors on their cellssuch as breast, endometrium, ovary cancers, hormones are used forpalliative therapy of the tumors. For example, tamoxifen, ananti-estrogen oral hormone, can bind to estrogen receptors on breastcancer cells and is used in palliative therapy of breast cancer, beingparticularly effective for metastatic breast cancer in thepostmenopausal woman. Therefore, the detection of the presence ofestrogen receptors in said tumor cells is a useful tool for theprognosis of the endocrine therapy.

Thus, in one embodiment, the invention further provides novel conjugatesof the peptides of the invention having estrogenic-like activity, suchas peptides 1 and 2, for detection of estrogen receptors on tumor cells,particularly breast cancer cells. The conjugates may be formed withfluorescent markers such as fluorescein isothiocyanate (FITC), in whichcase the estrogen receptors are detected in vitro by immunofluorescenceof breast cancer cells or tissue, or with chelating agents such aspentetic acid (DTPA) linked to either particles of a paramagneticelement such as gadolinium or of a radioactive element such as In⁺⁺⁺,for in vivo magnetic resonance imaging (MRI) or radioimmmunodetection,respectively, of breast cancer cells or tissue.

Thus the invention further provides an in vitro method for detection ofestrogen receptors in a cancer patient, particularly breast cancer, forprognosis of endocrine treatment of said patient, which comprisesreacting a suitable sample, such as a paraffin section, of tissue, e.g.breast tissue, obtained from said patient, with an estrogenic-likepeptide of the invention conjugated to a fluorescent marker such asfluorescein isothiocyanate (FITC), and detecting the estrogen receptorsby immumofluorescence of said tissue, the strong presence offluorescence in the nucleus of the cells of said tissue indicating thepresence of estrogen receptors.

In another embodiment, the invention further provides a method fordetection of estrogen receptors in a cancer patient, particularly breastcancer, for prognosis of endocrine treatment of said patient, whichcomprises injecting to said patient a conjugate of an estrogenic-likepeptide of the invention with DTPA and gadolinium, and performing MRI ofsaid patient, whereby the localization of gadolinium in the breasttissue indicates the presence of estrogen receptors.

In still another embodiment, the invention further provides a method fordetection of estrogen receptors in a cancer patient, particularly breastcancer, for prognosis of endocrine treatment of said patient, whichcomprises injecting to said patient a conjugate of an estrogenic-likepeptide of the invention with DTPA and a radioactive element such asIn⁺⁺⁺, and performing radioactive imaging of said patient, whereby thelocalization of the radioactive element in the breast tissue indicatesthe presence of estrogen receptors.

In addition, the invention further provides novel conjugates of theestrogenic peptides of the invention with chemotherapeutic drugs such asadriamycin and daunomycin, for affinity targeting and treatment ofestrogen-sensitive tumors, particularly breast cancer. The conjugatesallow localization of the chemotherapeutic drug and therapy with a loweramount of the toxic drug.

The peptides and peptide derivatives of the invention are obtained byany method of peptide synthesis known to those skilled in the art, suchas for example by solid phase peptide synthesis.

The present invention is also directed to pharmaceutical compositionscomprising a pharmaceutically acceptable carrier and at least onepeptide or peptide derivative of the invention having steroidhormone-like activity. The pharmaceutical composition will beadministered according to known modes of peptide administration,including oral, intravenous, subcutaneous, intraarticular,intramuscular, inhalation, intranasal, intrathecal, intradermal,transdermal or other known routes. The dosage administered will bedependent upon the age, sex, health condition and weight of therecipient, and the nature of the effect desired.

The peptides of the invention for use in therapy are typicallyformulated for administration to patients with a pharmaceuticallyacceptable carrier or diluent to produce a pharmaceutical composition.The formulation will depend upon the nature of the peptide and the routeof administration but typically they can be formulated for topical,parenteral, intramuscular, intravenous, intraperitoneal, intranasalinhalation, lung inhalation, intradermal or intra-articularadministration. The peptide may be used in an injectable form. It maytherefore be mixed with any vehicle which is pharmaceutically acceptablefor an injectable formulation, preferably for a direct injection at thesite to be treated, although it maybe administered systemically.

The pharmaceutically acceptable carrier or diluent may be, for example,sterile isotonic saline solutions, or other isotonic solutions such asphosphate-buffered saline.

The peptides of the present invention may be admixed with any suitablebinder(s), lubricant(s), suspending agent(s), coating agent(s),solubilising agent(s). It is also preferred to formulate the peptide inan orally active form.

Tablets or capsules of the peptides may be administered singly or two ormore at a time, as appropriate. It is also possible to administer thepeptides in sustained release formulations.

Typically, the physician will determine the actual dosage which will bemost suitable for an individual patient and it will vary with the age,weight and response of the particular patient. There can, of course, beindividual instances where higher or lower dosage ranges are merited,and such are within the scope of this invention.

Alternatively, the peptides of the invention, can be administered byinhalation or in the form of a suppository or pessary, or they may beapplied topically in the form of a lotion, solution, cream, ointment ordusting powder. An alternative means of transdermal administration is byuse of a skin patch. For example, they can be incorporated into a creamconsisting of an aqueous emulsion of polyethylene glycols or liquidparaffin. They can also be incorporated, at a concentration of between 1and 10% by weight, into an ointment consisting of a white wax or whitesoft paraffin base together with such stabilisers and preservatives asmay be required.

For some applications, preferably the compositions are administeredorally in the form of tablets containing excipients such as starch orlactose, or in capsules or ovules either alone or in admixture withexcipients, or in the form of elixirs, solutions or suspensionscontaining flavouring or colouring agents. For such oral administration,the peptide may preferably formed into microcapsules or nanoparticlestogether with biocompatible polymers such as polylactic acid and thelike.

The compositions (as well as the peptides alone) can also be injectedparenterally, for example intracavernosally, intravenously,intramuscularly or subcutaneously. In this case, the compositions willcomprise a suitable carrier or diluent. For parenteral administration,the compositions are best used in the form of a sterile aqueous solutionwhich may contain other substances, for example enough salts ormonosaccharides to make the solution isotonic with blood.

For buccal or sublingual administration the compositions may beadministered in the form of tablets or lozenges which can be formulatedin a conventional manner.

The peptides and peptide derivatives of the invention are for use in thetreatment of conditions in which steroid hormones are commonly used.

For example, the peptides having estrogenic-like activity may be usedfor treatment of hormone-dependent cancers such as breast, prostate andcolon cancer, in hormone-replacement therapy, in prevention and/ortreatment of osteoporosis, etc. In addition, these peptides may beuseful for the prevention and/or treatment of degenerative diseases ofthe central nervous system like Alzheimer's disease and Parkinson'sdisease as well as those resulting from trauma and stroke in the brain,based on recent findings that estrogens have neuroprotective effects inthe adult brain and exert beneficial effects in the treatment ofAlzheimer's disease and other neurodegenerative diseases of the centralnervous system like Parkinson's disease as well as those resulting fromtrauma and stroke in the brain (Wang et al., 2001; Munoz and Feldman,2000; Wise, 2000; Sapolsky and Finch, 2000; Green and Simpkins, 2000).Some of these peptides, unlike estradiol, recognize only ER α or ER β orboth receptors. Being more selective than estradiol, these peptides mayhave more clinical applications. For instance, estradiol is widely usedin hormone replacement therapy to help the maintenance of the CNS inpostmenopausal women. However, estrogen has unwanted side effects suchas an increased risk of uterine cancer. Since the mature uterus has verylittle ER β, peptides that are selective ER β agonists might beefficient in protecting the brain from age-related neurodegenerationwithout affecting the uterus.

As progestins, the peptides having progestational-like activity may beused for contraception or in endocrine therapy of breast cancer, uterinefibroids or polycystic ovary syndrome. As corticoid substitutes, thepeptides having adrenocorticoid-like activity may be used asanti-inflammatory in all disorders treated by corticoids.

The present invention further relates to a method of treatment of apatient suffering from a disorder that can be treated with a steroidhormone which comprises administering to said patient an effectiveamount of a peptide or peptide derivative of the invention. In specificembodiments, the method may be used for prevention and or treatment ofall conditions mentioned above.

The invention will now be illustrated by the following non-limitingExamples.

EXAMPLES

Materials and Methods

-   (i) Monoclonal Antibodies (mAbs): The preparation of specific mAbs    to steroids has been previously described (Kohen, 1986; Somjen,    1998). Anti-estradiol mAb clone 15 was derived from a BALB/c female    mouse immunized with estradiol-6-(O-carboxymethyl)oxime-bovine serum    albumin (E2-6.BSA). Clone 15 belonged to the IgG1 class. Purified    IgG fraction was prepared by affinity chromatography on    Sepharose-Protein A as previously described (Strassburger and Kohen,    1990). The mAb to progesterone, clone 1E11, was obtained from a CD2    mouse immunized with progesterone-11-alpha hemisuccinate-BSA as    previously described (De Boever et al., 1989).-   (ii) Biotinylation of anti-estradiol clone 15: Anti-estradiol IgG,    clone 15 (100 μg) in NaHCO₃ (100 μl) was reacted with 5 μl of    N-hydroxysuccinimide ester of biotin solution in dimethylfomiamide    (1 mg/ml) for 2 hours at room temperature. The reaction mixture was    then dialysed at 4° C. against phosphate-buffered saline (PBS, pH    7.4). Biotinylation was ensured by binding of the biotinylated mAb    on streptavidin-coated plates.-   (iii) Screening for monoclonal anti-estradiol (anti-E2) binding    phages: The phage-peptide library (a 15-mer library obtained    from J. J. Devlin, Cetus Corporation, Emeryville, Calif., USA), was    subjected to three rounds of affinity selection against the    biotinylated mAb to estradiol, clone 15, as described before    (Devlin J. J., 1990), with minor modifications. Petri plates (60 mm,    Nunc) were coated with 800 μl streptavidin (10 μg/ml in 0.1M NaHCO₃,    pH 9.6) for overnight at 4° C. and then blocked with PBS containing    3% BSA and 0.1 mg/ml streptavidin 1 hour at 37° C. An aliquot of the    15-mer library containing 1×10¹¹ phage particles was incubated with    2.5 μg biotinylated clone 15 in 40 μl PBS containing 0.5% BSA for    overnight at 4° C. The streptavidin-coated plates were washed 6    times with PBS containing 0.05% Tween-20 (PBST). The preincubated    phage-mAb mixture was diluted to 600 μl with PBST and then    transferred to the streptavidin-coated plate. Following incubation    for 30 minutes at room temperature with gentle shaking, the plates    were washed 10 times with PBST-0.05, over a period of 1 h. Bound    phages were eluted with 600 μl 0.1 M glycine.HCl (pH 2.2) containing    1 mg/ml BSA for 10 min and immediately transferred to 50 μl 2 M    Tris. An aliquot of the eluate was retained for titration and the    rest of the eluate was amplified by infecting log phase culture of    E.coli K91 Kan and plated on 150 mm plates LB containing 100 μg/ml    ampicillin. After incubation overnight at 37° C., the cells were    harvested in 10 ml PBS and centrifuged. The amplified phages in the    supernatant were purified by precipitating twice with polyethylene    glycol (PEG)/NaCl (Smith and Scott, 1993). The final phage pellet    obtained was resuspended in 500 μl PBS, quantified    spectrophotometrically and used for subsequent rounds of panning. In    order to select high affinity clones the amount of biotinylated mAb    was progressively reduced to 1 μg and 0.1 μg in the second and third    rounds of panning, respectively. Washing steps were performed with    PBS containing 0.5% Tween 20 in the second and third panning.

For titration, 10 μl of an appropriate dilution of the phage wasincubated with 100 μl of log phase culture of K91 Kan cells for 10 minat 37° C. and spread on LB plates containing 100 μg/ml of ampicillin.

-   (iv) Selection of anti-E2 mAb binding phages by biopanning: Well    isolated ampicillin-resistant colonies from the third round of    panning were individually inoculated into 150 μl of superbroth    containing 100 μg/ml of ampicillin in 96-well tissue culture plates    (Costar). After incubation overnight at 37° C., the cells were    pelleted and the phage-supernatants transferred to another 96-well    tissue culture plate and stored at 4° C.

For phage-ELISA, microtiter plates (Maxisorb, Nunc, Neptune, N.J.) werecoated overnight at 4° C. with 50 μl rabbit anti-M13 antiserum (1:2000dilution in 0.1M NaHCO3). Plates were then washed thrice with PBS-0.05Tand incubated with 3% blocking buffer (1.5% BSA and 1.5% hemoglobin inPBS) for 1 h at 37° C. Following three washes with PBS-0.05T, 50 μl ofphage supernatants (obtained as described above) from each clone werethen added to individual wells in the ELISA plate. The plate wasincubated for 1 h at 37° C. to capture the phages. The plate was washedthree times with PBS-0.05T and incubated with biotinylatedanti-estradiol mAb (diluted to 1 μg/ml in 1% blocking buffer) forovernight at 4° C.

-   (v) Sequencing of phage DNA: Phages from selected clones were    purified by PEG/NaCl precipitation, single-stranded DNA was prepared    and sequences were determined by a dideoxy chain termination method    using an Applied Biosystems (Perkin Elmer) sequenator.-   (vi) Synthetic peptides: Peptides were synthesized by the Chemical    Services Unit or at the Department of Organic Chemistry of the    Weizmann Institute of Science, Rehovot, or at the Hebrew University,    Jerusalem, both in Israel, and were purified by known methods.-   (vii) Screening for monoclonal anti-progesterone (anti-P) binding    phages. Biopanning for anti-P binding phages was performed    essentially as described for the screening of anti-E2 binding phages    (in (iv) above) with the following modifications: A solution of    purified anti-P mAb IgG, clone 1E11 (100 μg/ml in 0.1 M NaHCO_(3,)    100 μl) was added to two wells of a 96 well-microtiter plate and    incubated for overnight at 4° C. The unbound solution was discarded    and the plates were blocked with PBS containing 3% BSA 1 h at 37° C.    Meanwhile, an aliquot of the original peptide library (15-mer linear    library from Devlin or 16-mer constrained library from Smith, 10¹¹    phage particles) was incubated with the blocking solution in a total    volume of 225 μl for 2 h at 37° C. The wells coated with anti-P mAb    were washed 6 times with PBS-0.1T and 100 μl. of preincubated phage    library was added to each well. After 4 hours incubation at room    temperature, the contents were discarded. The wells were filled with    PBS-0.1T, shaken for a minute and the contents were discarded. In    the same way, washing was repeated 10 times, following which the    bound phages were eluted with 0.1 M glycine.HCl (pH 2.2) containing    1 mg/ml BSA (100 μl /well) for 10 min and immediately transferred to    tubes containing 9 μl 2 M Tris. Eluates obtained using each library    were individually pooled. Amplification and purification of the    phages were done as explained for anti-E2 binding phages. In order    to select high affinity clones the coating concentration of mAb was    reduced to 1 μg and 0.1 μg per well in the second and third rounds    of panning, respectively. The rest of the steps were performed as    described for anti-E2 mAb.

Example 1

Selection of Phages and Peparation of Synthetic Peptides

As described above in Materials and Methods, biotinylated anti-E2 clone15 was incubated with a 15-mer phage epitope library, and the phage-mAbcomplexes were captured on streptavidin-coated plates. Bound phages wereeluted with 0.1M HCl.glycine pH 2.2, amplified and used for subsequentrounds of panning. After three rounds of panning, phage clones werescreened for mAb binding by phage-ELISA. Thirty positive clones wereselected and the DNA encoding the displayed peptide were sequenced.

The insert sequences represented three different peptide sequences asshown in Table 1. TABLE 1 Peptides selected from a 15-mer phage displaypeptide library using anti-estradiol mAb, clone 15, as probe.Sequence^(a) Frequency^(b) H5 LPALDPTKRWFFETK 21 G5 AHWNSENTVVGLPSK 6 H8GMQMHQRHVYLSKRP 3^(a)Amino acid sequences of peptide inserts were deduced from DNAsequencing of the insert of the positive phage clones.^(b)Frequency denotes the number of phage clones with identical inserts.

There was no consensus among the three selected peptides. Initialbinding studies indicated that only peptide H5, herein in thespecification and claims identified as peptide 1, recognized anti-E2 mAbclone 15 (see below). Accordingly, we concentrated our studies on thislinear peptide 1.

The molecular weight of the linear peptide 1 was confirmed by electronmass spectrometry as being of 1848 (M-1).

Example 2

Synthesis of the Extended Cyclic Peptide 2

Based on our earlier studies, we found that the affinity of the peptidesselected from phage display library can be significantly improved byincorporating flanking aminoacids of the phage coat protein andsubsequent cyclization (Venlcatesh et al., 2000), at least for fewepitopes which are conformationally constrained. In order to checkwhether the same is true with the linear peptide 1, we synthesized aderivative, herein designated peptide 2, by flanking peptide 1 withresidues -AEC at the N-terminus and -PPPPC at the C-terminus, and airoxidized the peptide to prepare a cyclized peptide 2 having thesequence:

For the synthesis of peptide 2, the extended linear peptideCAELPALDPTKRWFFETKPPPPC (10 mg) was air-oxidized by stirring a 0.25mg/ml solution of the peptide in water, pH 10.00 (adjusted with ammoniumhydroxide) for 48 h at room temperature. Complete oxidation was ensuredby estimating the lack of free-SH using Ellman's reagent. Formation of asingle monomeric cyclic peptide was ensured by HPLC and by molecularweight determination using electron mass spectrometry. The cyclizedpeptide 2 showed a molecular weight of 2643 (M−1).

Example 3

Preparation of Fluorescent Labeled Linear Peptide 1

The linear peptide 1 of the sequence: LPALDPTKRWFFETK contains 2 lysine(K) and 1 glutamic acid (E) residues and can be easily conjugated toseveral molecules of interest.

For the conjugation with fluorescein, the linear peptide 1 (1.8 mg) wasdissolved in 200 μl of 50 mM carbonate-bicarbonate buffer (pH 9.6).Celite-FITC powder (6.4 mg) was added and the reaction mixture wasstirred overnight at room temperature and subsequently centrifuged. Thefiltrate was purified by gel filtration on Sephadex G-10 column using 50mM Tris-HCl, pH 7.45 as eluant. UV measurement of the eluate at 495 nmindicated that 2 moles FITC were incorporated per mole of the peptide.The labeled peptide was stored at −20° C. in the dark until use. Thisfluorescein labeled peptide 1 displayed the same binding activity toanti-E2 clone 15 as the original linear peptide 1 (not shown).

Example 4

Characterization of the Peptides in Terms of Antibody Recognition

ELISA methodology was used first for the initial screening of thevarious peptides for antibody binding specificity. In this system, theanalyte (e.g. estradiol, testosterone, progesterone, estriol) or thepeptides and a defined amount of the homologous solid-phasehapten-protein conjugate [e.g. estradiol-6-(O-carboxymethyl) oximeovalbumin, testosterone-3-(O-carboxymethyl)oxime ovalbumin,progesterone-11a-hemisuccinate ovalbumin orestriol-6-(O-carboxymethyl)oxime ovalbumin] compete for a limitedconcentration of the specific homologous antibody. The linear peptide 1and the cyclized peptide 2 at a concentration of 0.5 mM inhibited by 36%the binding of immobilized estradiol ovalbumin conjugate to anti-E2,clone 15, the antibody used in the screening of the phage displaypeptide library, but not of other high affinity anti-E2 antibodies,clones 8D9, 11B6 and 2F9 (Somjen et al., 1998). On the other hand, E2used as a control at a concentration of 150 nM inhibited by 30% thebinding of estradiol ovalbumin conjugate of all the anti-E2 antibodiesthat were tested. Moreover, the peptides were not capable of competingwith the various solid phase hapten protein conjugates for the bindingsites of the homologous anti-steroidal antibodies (eg anti-progesterone,clone 1E11, anti-estriol, clone 9B4, anti-testosterone, clone 5F2).These results indicate that the linear peptide 1 isolated from the phagepeptide display library recognizes an epitope of only anti-E2 clone 15and not of other high affinity anti-steroidal antibodies (data notshown).

Example 5

The Synthetic Peptides 1 and 2 Compete With Estrogen for the BindingSites of Anti-E2 Clone 15 in a Competititve Immunoassay System UsingRadioactivity or Fluorescence as an End Point.

In order to confirm the specific binding activity of the peptides 1 and2 for anti-E2 mAb, clone 15, a radioimmunoassay system or atime-resolved fluoroimmunoassay system were used where the peptidescompeted with [³H]-estradiol or with estradiol ovalbumun europiumconjugate for the binding sites of clone 15. Dose response curves forestradiol and the peptides using radioimmunoassay are shown in FIG. 1.The two synthetic peptides 1 and 2 competed with [³]H-estradiol for thebinding sites of the anti-estradiol mAb, clone 15, with an IC₅₀ of about5 μM whereas the IC₅₀ for estradiol was <0.8 nM. In contrast to ourearlier observation (Venkatesh et al., 2000), we did not observe anyimprovement in the binding affinity of the cyclic peptide 2 towardsanti-E2 15.

Example 6

Synthetic Peptides 1 and 2 Bind Estradiol Receptor (ER) α but not ER β

The dose response curves of estradiol and of the linear and cyclicpeptides 1 and 2 for binding to ER α and β are shown in FIGS. 2 and 3,respectively. The binding affinity of estradiol for the two receptorswas high (IC₅₀ 1 nM). On the other hand, unlike estrogen, the twopeptides showed selectivity for binding to ER α, but none to ER β.Interestingly, the cyclic peptide 2 had a relatively better affinity toER α (IC₅₀ 100 μM) as compared to the linear peptide 1 (IC₅₀ 500 μM),suggesting that the binding of the ligand to the ER is conformationdependent.

Example 7

The Synthetic Peptides 1 and 2 Induce Creatine Kinase Activity in RatTissues in Vivo

Treatment of immature female rats for 4 hours with estradiol (5 μg/rat)or the cyclic peptide 2 (0.5 mg/rat) caused an increase in the specificactivity of creatine kinase (CK) in all the tissues (uterus, aorta, andleft ventricle) that were examined. On the other hand the linear peptide1 at this concentration (0.5 mg/rat) caused an increase in CK activityonly in the uterus and left ventricle. At higher concentration (2.5mg/rat) the linear peptide 1 stimulated the CK activity in the epiphysisand aorta as well (data not shown). When rats were treated with acombination of the estrogen receptor antagonist raloxifene withestradiol or with the peptides 1 and 2, the increase in CK activitycould be blocked in all the tissues with the exception of the epiphysiswhere raloxifene could not inhibit the stimulatory activity of thecyclic peptide 2.

Immature female rats were injected with estradiol (5 μg/rat), the cyclicpeptide 2 (0.5 mg/rat), the linear peptide 1 (0.5 mg/rat), raloxifene(0.5 mg/rat), the cyclic peptide 2 (0.5 mg/rat) plus raloxifene (0.5mg/rat), the linear peptide 1 (0.5 mg/rat) plus raloxifene (0.5 mg/rat)or estradiol (5 μg/rat) plus raloxifene (0.5 mg/rat). The control goupsreceived 0.5 ml saline containing 0.5% ethanol or tris-saline alone. Thevarious organs were assayed for CK activity four hours after treatment.The results are shown in Table 2 and are expressed as means ±S.E.M., forn=15, and further expressed as experimental (E) over control (C) wherethe control is given a value of 1.0. TABLE 2 Stimulation of the specificactivity of creatine kinase by estrogen and synthetic peptides 1 and 2in rat tissues in vivo Creatine kinase specific activity(Experimental/control) Organ Treatment Uterus Diapysis Epipysis AortaLV# Control   1 ± 0.13   1 ± 0.19   1 ± 0.18   1 ± 0.13   1 ± 0.2Estradiol 1.37 ± 0.07** 1.87 ± 0.1* 1.37 ± 0.07**  2.0 ± 0.18* 1.83 ±0.12* Peptide 1  1.6 ± 0.27* 1.09 ± 0.17 1.10 ± 0.09 1.35 ± 0.23 1.41 ±03** Peptide 2 1.36 ± 0.11** 1.39 ± 0.10** 1.37 ± 0.08** 1.79 ± 0.12*1.61 ± 0.15** Raloxifene 0.99 ± 0.17 1.75 ± 0.13* 1.41 ± 0.05* 1.63 ±0.04* 1.34 ± 0.11** Raloxifene + peptide 1 0.92 ± 0.05 1.21 ± 0.18 1.04± 0.14 1.18 ± 0.18 1.12 ± 0.19 Raloxifene + peptide 2 1.24 ± 0.12 1.37 ±0.31 1.56 ± 0.07* 1.36 ± 0.24 0.91 ± 0.20 Raloxifene + estradiol 1.20 ±0.11 0.78 ± 0.20 1.14 ± 0.16 1.41 ± 0.36 0.75 ± 0.21(Walter et al., 1985)*p < 0.01;**p < 0.05; treated vs control#LV = left ventricle

Example 8

Peptides Obtained from the Linear Peptide 1 by Deletion of One or MoreAmino Acid Residues and Their Properties

The linear peptide 1 is 15-amino acid long. We further explored thepossibility whether smaller peptides derived from peptide 1 may haveantibody binding activity and receptor binding activity. In order tofind out the minimum amino acid sequence required in the linear peptide1 for antibody and receptor binding, several peptides with varyinglength of from 4 to 14 amino acids long were synthesized, and were firstevaluated in terms of binding to anti-estradiol (E2) mAb clone 15. Thepeptides showing similar or better antibody binding activity than thelinear peptide 1 were further evaluated in terms of binding to ERα andβ. Table 3 shows the results. TABLE 3 Peptides derived from peptide 1Inhibition Inhibition of binding of binding Inhibition of of labeled oflabeled binding of E2* to E2** to labeled E2** Peptide Sequence Anti-E2ERα to ERβ  1 LPALDPTKRWFFETK + + −  7 LPALDPTKRWFFET + N.E. N.E.  9LPALDPTKRWFEE + N.E. N.E. 15 LPALDPTKRWFF − N.E. N.E. 16 LPALDPTKRWF −N.E. N.E.  6  PALDPTKRWFFETK + N.E. N.E.  8   ALDPTKRWFFETK + N.E. N.E.14    LDPTKRWFFETK + N.E. N.E. 17     DPTKRWFFETK + N.E. N.E. 18     PTKRWFFETK + N.E. N.E. 35       TKRWFFETK + N.E. N.E. 42     PTKRWFFE + N.E. N.E. 43       TKRWFFE + N.E. N.E. 44       KRWFFE + + + 45         RWFFE + − N.E. 46          WFFE + − N.E.*labeled E2 = [³H]-Estradiol; **labeled E2 = estradiol ovalbumineuropium conjugate;N.E = not evaluated

The results shown in Table 3 indicate that a tetrapeptide with sequenceof WFFE is the minimal length required for inhibition of binding ofestradiol ovalbumin europium conjugate to anti-estradiol mAb clone 15.However, the tetrapeptide WFFE did not show any inhibition of binding of[³H]-estradiol to the estrogen receptors. On the other hand, thehexapeptide designated 44, of the sequence: KRWFFE, unlike the linearpeptide 1, recognized estrogen receptor α as well as β.

Since peptide 44 showed binding activity to the estrogen receptors, weproceeded to evaluate the amino acid residues that are necessary forbinding to the estrogen receptors by alanine screen. Six peptides inwhich every amino acid residue in peptide 44 was replaced by alaninewere synthesized and evaluated for antibody binding activity and, thosethat were positive, also for receptor binding activity. Table 4 showsthe results. TABLE 4 Properties of peptides obtained from peptide 44(KRWFFE) by alanine screen Inhibition of Inhibition of Inhibition ofbinding of binding of binding of labeled E2 to labeled E2 to labeled E2to Peptide Sequence anti-E2 ERα ERβ A-43 ARWFFE + + − A-44 KAWFFE + + +A-45 KRAFFE − A-46 KRWAFE − A-47 KRWFAE − A-48 KRWFFA −

The results shown in Table 4 indicate that only two peptides (A-43 andA-44) showed inhibition of binding of estradiol ovalbumin europiumconjugate to anti-estradiol mAb clone 15. When the estrogen bindingability of these two peptides was evaluated, peptide A-44 recognizedboth estrogen receptor α and β whereas peptide A-43 recognized only theestrogen receptor α and not β. These results indicate that the lysineresidue K at position 1 of the 6-mer peptide is essential for binding toestrogen receptor α and not to estrogen receptor β and the arginineresidue R at position 2 of the 6-mer peptide is not essential forbinding to estrogen receptor α or β. Moreover, amino acid residues WFEEare essential for binding to anti-estradiol mAb clone 15.

Since peptide 44 showed estrogen receptor binding activity and betterinhibition of estradiol ovalbumin europium to anti-estradiol mAb clone15 than the linear peptide 1, several peptides were synthesized in whicheach amino acid residue in peptide 44 was replaced by another amino acidresidue belonging to the same group of amino acids (e.g. charged,hydrophobic, aromatic, polar, positive, aliphatic, small and tiny).Table 5 shows the results obtained with the peptides showing estrogenlike activity. TABLE 5 IC₅₀ to IC₅₀ to Peptide Sequence Anti-E2 15 ER αIC₅₀ to ERβ Linear 1 LPALDPTKRWFFETK 1000 nM  500 μM none Cyclic 2CAELLPALDPTKRWFFETKPPPPC 1000 nM  100 μM none 44 KRWFFE 35 nM 400 μM 400μM A43 ARWFFE 40 nM 500 μM None A44 KAWFFE 40 nM 250 μM 250 μM 39 VRWFFE15 nM <100 μM   <100 μM   19 KSWFFE 40 nM None None 21 KPWFFE 45 nM 500μM >500 μM   B33 VPWFFE  6 nM None None B34 VSWFFE  6 nM 250 μM 100 μMB35 VRWFYE  6 nM None None B37 IRWFFE 20 nM None 250 μM B38 LRWFFE 40 nMNone None

The results shown in Table 5 indicate that peptides 44, A43, A44, 39,19, 21, B33, B34, B35, B37, B38 inhibit one and half order of magnitudebetter than the linear or cyclic peptides 1 and 2 the binding ofestradiol ovalbumin europium conjugate to anti-E2 mAb clone 15. Unlikethe cyclic peptide 2, peptides 44, A44, 39, 21, B34 recognized bothestrogen receptors. Interestingly, peptide B37 recognized only the ERβand peptides 19, B33, B35 and B38 did not show any binding activity tothe estrogen receptors.

When the biological activity of peptide 39 was evaluated in vivo, thispeptide stimulated the specific activity of creatine kinase in rattissues (uterus, left ventricle, aorta, diaphysis, and epiphysis) at adose of 100 μg/rat, a dose 5 times lower than the cyclic peptide 2 (notshown).

Example 9

The Synthetic Progestational-like Peptides 3 and 4 Compete withProgesterone for the Binding Sites of Anti-progesterone Clone 1E11 in aRIA System

In order to confirm the specific binding activity of the peptides 3 and4 for the anti-progesterone mnAb clone 1E11, a RIA system where thepeptides competed with [³H]-progesterone for the binding sites of clone1E11 was used. Dose response curves for progesterone and the twosynthetic peptides are shown in FIG. 4. The peptide 4 competed with[³H]-progesterone for the binding sites of the anti-progesterone clone1E11 with IC₅₀ of about 5 μM whereas the IC₅₀ of progesterone itself was<1 nM. On the other hand, the IC₅₀ of peptide 3 was about 100 μM.

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1. (Cancelled)
 2. A synthetic peptide that mimics the biologicalactivity of a steroid hormone, said synthetic peptide being selectedfrom the group consisting of: (i) a peptide exhibiting a steroidhormone-like biological activity; (ii) a peptide obtained from (i) bydeletion of one or more amino acid residues; (iii) a peptide obtained byaddition to a peptide (i) or (ii) of one or more natural or non-naturalamino acid residues; (iv) a peptide obtained by replacement of one ormore amino acid residues of a peptide (i) to (iii) by the correspondingD-stereomer, by another natural amino acid residue or by a non-naturalamino acid residue; (v) a chemical dervative of a peptide (i) to (iv);(vi) a cyclic derivative of a peptide (i) to (v); (vii) a dual peptideconsisting of two of the same or different peptides (i) to (vi), whereinthe peptides are covalently linked to one another directly or through aspacer; and (viii) a multimer comprising a number of the same ordifferent peptides (i) to(vi).
 3. The peptide according to claim 2,wherein said peptide has estrogenic-like activity and mimics theactivity of an estrogen selected from the group consisting of estradiol,estrone or estriol.
 4. (Cancelled)
 5. The peptide according to claim 3,wherein said estrogen is estradiol and said peptide havingestrogenic-like activity has at least 6 amino acid residues of thesequence:X₄-X₂-Trp-Phe-X₁-Glu-X₃ wherein X₁ is Phe or Tyr; X₂ is Lys-Arg-,Ala-Arg, Lys-Ala-, Val-Arg-, Lys-Pro-, Val-Ser-, or Ile-Arg-; X₄ ishydrogen or Thr-, Pro-Thr-, Asp-Pro-Thr-, Leu-Asp-Pro-Thr-,Ala-Leu-Asp-Pro-Thr-, Pro-Ala-Leu-Asp-Pro-Thr-, Leu-Pro-Ala-LeuAsp-Pro-Thr-, or Cys-Ala-Glu-Leu-Pro-Ala-Leu-Asp-Pro-Thr; and X₃ ishydroxyl, Thr, -Thr-Lys, or -Thr-Lys-Pro-Pro-Pro-Pro-Cys; and cyclicderivatives thereof.
 6. The peptide according to claim 5, selected fromthe group consisting of the peptides:Leu-Pro-Ala-Leu-Asp-Pro-Thr-Lys-Arg-Trp-Phe- (1) Phe-Glu-Thr-Lys Cyclic[Cys-Ala-Glu-Leu-Pro-Ala-Leu-Asp-Pro- (2)Thr-Lys-Arg-Trp-Phe-Phe-Glu-Thr-Lys-Pro-Pro- Pro-Pro-Cys]Lys-Arg-Trp-Phe-Phe-Glu (44) Ala-Arg-Trp-Phe-Phe-Glu (A43)Lys-Ala-Trp-Phe-Phe-Glu (A44) Val-Arg-Trp-Phe-Phe-Glu (39)Lys-Pro-Trp-Phe-Phe-Glu (21) Val-Ser-Trp-Phe-Phe-Glu (B34)Ile-Arg-Trp-Phe-Phe-Glu (B37)


7. A peptide according to claim 2, wherein said peptide hasprogestational-like activity and mimics the activity of progesterone. 8.(Cancelled)
 9. The peptide according to claim 7, wherein said peptidehaving progesterone-like activity is selected from the group consistingof the peptides 3 and 4, of the sequences:Val-Asn-His-Pro-Trp-Asp-Gln-Ala-Gln-Phe-Leu- (3) Ser-Thr-IleSer-Asn-Pro-Phe-Cys-Gln-Thr-Asp-Gly-Asp-Cys- (4) His-Val-His-Thr


10. A peptide according to claim 2, wherein said peptide hasandrogenic-like activity and mimics the activity of testosterone. 11.(Cancelled)
 12. A peptide according to claim 2, wherein said peptide hasadrenocorticoid-like activity and mimics the activity of anadrenocorticoid hormone selected from the group consisting of cortisone,hydrocortisone or corticosterone.
 13. (Cancelled)
 14. A pharmaceuticalcomposition comprising a peptide according to claim 2, and apharmaceutically acceptable carrier.
 15. A pharmaceutical compositioncomprising a peptide according claim 3, and a pharmaceuticallyacceptable carrier for the purpose of estrogen replacement therapy. 16.A pharmaceutical composition according to claim 15, for treatment ofhormone-dependent cancers selected from the group consisting of breast,prostate and colon cancer, postmenopausal symptoms or prevention and/ortreatment of osteoporosis.
 17. A pharmaceutical composition according toclaim 15, for prevention and/or treatment of degenerative diseases ofcentral nervous system.
 18. A pharmaceutical composition according toclaim 17, for prevention and/or treatment of Alzheimer's disease,Parkinson's disease or another degenerative disease of the centralnervous system resulting from trauma or stroke in the brain.
 19. Apharmaceutical composition comprising a peptide according to claim 7,and a pharmaceutically acceptable carrier for the purpose ofcontraception or in endocrine therapy of breast cancer, uterine fibroidsor polycystic ovary syndrome.
 20. (Cancelled)
 21. (Cancelled) 22.(Cancelled)
 23. (Cancelled)
 24. (Cancelled)
 25. (Cancelled) 26.(Cancelled)
 27. (Cancelled)
 28. (Cancelled)
 29. A method for screening acombinatorial peptide library for the identification of a peptideexhibiting a steroid hormone biologic-like activity, which comprises:(i) providing a monoclonal antibody with high affinity and specificityto the steroid hormone investigated; (ii) screening a combinatorialpeptide library with said monoclonal antibody of (i) for theidentification of peptides that bind specifically to said monoclonalantibody; (iii) isolating said peptides and testing them for competitionwith said steroid hormone for binding to said monoclonal antibody invitro and for binding to said steroid hormone receptor in vitro; and(iv) identifying the peptides that mimic said steroid hormone activityas being those that successfully compete with said steroid hormone forbinding to its monoclonal antibody and receptor.
 30. A conjugate of apeptide according to claim 2 with a label selected from the groupconsisting of a fluorescent, a paramagnetic and a radioactive marker.31. A conjugate according to claim 30, wherein said peptide is a peptidethat has estrogen-like activity and that mimics the activity of anestrogen selected from the group consisting of estradiol, estrone orestriol.
 32. A conjugate of a peptide according to claim 2 with achemotherapeutic drug.
 33. A conjugate of a peptide according to claim 3with a chemotherapeutic drug.